L1, a novel target of beta-catenin signaling, transforms cells and is expressed at the invasive front of colon cancers

Aberrant beta-catenin-TCF target gene activation plays a key role in colorectal cancer, both in the initiation stage and during invasion and metastasis. We identified the neuronal cell adhesion molecule L1, as a target gene of beta-catenin-TCF signaling in colorectal cancer cells. L1 expression was high in sparse cultures and coregulated with ADAM10, a metalloprotease involved in cleaving and shedding L1's extracellular domain. L1 expression conferred increased cell motility, growth in low serum, transformation and tumorigenesis, whereas its suppression in colon cancer cells decreased motility. L1 was exclusively localized in the invasive front of human colorectal tumors together with ADAM10. The transmembrane localization and shedding of L1 by metalloproteases could be useful for detection and as target for colon cancer therapy.     

Epithelial-mesenchymal transition and the invasive potential of tumors

The development of metastasis requires the movement and invasion of cancer cells from the primary tumor into the surrounding tissue. To acquire such invasive abilities, epithelial cancer cells must undergo several phenotypic changes. Some of these, including alterations in cell adhesion and migration, are reminiscent of those observed during the developmental process termed epithelial-mesenchymal transition (EMT). Several master gene regulatory programs known to promote EMT during development have recently been discovered to play key roles in cancer progression. In particular, the regulation of cell adhesion molecules and the signaling pathways linking them to mechanisms of gene regulation has emerged as an important determinant of tumor cell invasion and metastasis. A deeper understanding of these mechanisms should allow both better diagnosis and the development of specific treatments for invasive cancer.     

Autoregulation of actin synthesis responds to monomeric actin levels

Regulation of the assembly and expression of actin is of major importance in diverse cellular functions such as motility and adhesion and in defining cellular and tissue architecture. These biological processes are controlled by changing the balance between polymerized (F) and soluble (G) actin. Previous studies have indicated the existence of an autoregulatory pathway that links the state of assembly and expression of actin, resulting in the reduction of actin synthesis after actin filaments are depolymerized. We have employed the marine toxins swinholide A and latrunculin A, both disrupting the organization of the actin-cytoskeleton, to determine whether this autoregulatory response is activated by a decrease in the level of polymerized actin or by an increase in monomeric actin concentrations in the cell. We showed that in cells treated with swinholide A the level of filamentous actin is decreased, and using a reversible cross-linking reagent, we found that actin dimers are formed. Latrunculin A also disassembled actin filaments, but produced monomeric actin, followed by a reduction in actin and vinculin expression, while swinholide A treatment elevated the synthesis of these proteins. In cells treated with both latrunculin A and swinholide A, dimeric actin was formed, and actin and vinculin synthesis were higher than in control cells. These results suggest that the substrate that confers an autoregulated reduction in actin expression is monomeric actin, and when its level is decreased by dimeric actin formation, actin synthesis is increased. J. Cell. Biochem. 65:469–478. © 1997 Wiley-Liss Inc.     

Cell density and cell shape-related regulation of vimentin and cytokeratin synthesis : Inhibition of vimentin synthesis and appearance of a new 45 kD cytokeratin in dense epithelial cell cultures

The pattern of the intermediate type filament protein synthesis was examined in cultured bovine mammary gland epithelial (BMGE) cells under conditions of varied cell shape and cell-cell contact. In dense monolayer and suspension cultures BMGE cells expressed a new cytokeratin of 45 kD identified as a member of the acidic subfamily of cytokeratins. This polypeptide has a phosphorylated component and is dissociated from the cytokeratins complex in the presence of 6.5 M urea. The mRNA of the new cytokeratin accumulated in dense cell cultures, as revealed by in vitro translation in a cell-free system. In BMGE-H cells that express also vimentin, the synthesis of vimentin decreased dramatically in dense cell cultures, while the synthesis of the 45 kD cytokeratin was maximal under these conditions. The results suggest that the expression of certain cytokeratins and that of vimentin can be coordinately regulated by factors in the cellular environment that effect cell shape and cell surface contacts.     

Tumor promoter-induced disruption of junctional complexes in cultured epithelial cells is followed by the inhibition of cytokeratin and desmoplakin synthesis

The organization and synthesis of proteins involved in the formation and stabilization of desmosome-type junctions was investigated in cultured epithelial cells treated with a tumor promoter (12-O-tetradecanoyl-phorbol-13-acetate (TPA]. In Madin-Darby bovine (MDBK) and canine (MDCK) kidney cell colonies, TPA induced a rapid disruption of desmosomes and marked alterations in cell morphology. Within 4-6 h after TPA treatment, cell shape changed from cuboidal to highly irregular, with some very long extensions that contained cytokeratin fibrils, and many flat lamellar protrusions which were devoid of cytokeratin fibrils. These morphological changes in both MDBK and MDCK cells were followed by a dramatic and coordinated inhibition in the synthesis of all cytokeratins, 14-24 h after the addition of TPA, but without a similar effect on the synthesis of vimentin, which is coexpressed in these cells. In contrast, in dense cultures of MDBK and MDCK cells the synthesis of cytokeratins and the organization of desmosomal contacts were not affected by TPA. In an epithelial cell line derived from the bovine mammary gland (BMGE-H) the synthesis of an acidic cytokeratin of 45 kD, which was previously shown to be synthesized at high levels only in dense cultures, was dramatically inhibited by TPA treatment. Cell-free in vitro translation assays with mRNA from control and TPA-treated cells also demonstrated a decrease in the synthesis of cytokeratins in response to TPA. The inhibition of cytokeratin synthesis after TPA treatment was paralleled by a decrease in the synthesis of a high molecular weight (HMW) desmoplakin protein, which was abundant in dense MDBK and BMGE-H cells. The results with TPA-treated cells are suggestive of a coordinated down-regulation in the synthesis of only those cytokeratins and of a desmoplakin which were shown to be regulated by the extent of cell-cell contact. Cytokeratin phosphorylation in TPA-treated cells was low and reflected the decrease in their total mass, suggesting that it was not altered by TPA treatment. The possible linkage between the regulation of synthesis and organization of proteins involved in desmosome formation is discussed.     

Evaluation of Seven Function-Known Candidate Genes for their Effects on Improving Drought Resistance of Transgenic Rice under Field Conditions

Many stress responsive genes have been reported with an effect on improving stress resistance in model plants under greenhouse conditions. Towards identification of genes for drought resistance breeding, seven well documented genes （CBF3, SOS2, NCED2, NPK1, LOSS, ZAT10, and NHX1） in stress resistance were selected in this study and transformed into rice cultivar Zhonghua 11 under the control of constitutive promoter Actinl and stress-inducible pro- moter of a rice HVA22 homolog, and transgenic rice were tested for drought resistance under field conditions. A total of 1598 independent transgenic To plants were generated. The percentages of single copy and expression of the transgenes were 36.7% and 57.6%, respectively. For each gene construct, 30 T1 families with expression of transgene were selected for drought resistance testing at the reproductive stage in field, and 10 of them were tested in PVC pipes with a defined stress protocol at the same stage. Relative yield and relative spikelet fertility were used as two major criteria to evaluate drought resistance performance because significantly decreased yield was observed in the T1 generation, Trans- genic families of eight constructs （HVA22P：CBF3, HVA22P：NPK1, Actin 1：LOS5, HVA22P：L OS5, Actin 1：ZA T10, HVA22P：ZA T10, Actinl：NHX1, and HVA22P：NHX1） showed significantly higher RY than wild-type （WT） under both drought stress field and PVC tube conditions. Transgenic families of 9 constructs （HVA22P.SOS2 and CBF3, LOS5, ZAT10, and NHX1 by both promoters） showed significantly higher relative spikelet fertility than WT in the field or PVC pipes. In the field drought resistance testing of T2 families derived from the T1 families with relatively lower yield decrease, transgenic families of seven constructs （HVA22P：CBF3, Actinl：NPK1, HVA22P：NPK1, Actinl：LOS5, HVA22P：LOS5, Actin1：ZAT10, and HVA22P：ZAT10） showed significantly higher yield per plant than WT, and families of nine constructs （Actinl：CBF3, HVA22P：CBF3,     

Isolation of a new polymorphic TC maize microsatellite located on the long arm of chromosome 10

Simple sequence repeats (SSR), also called microsatellites, were previously proved to be an important class of DNA markers. The isolation, mapping and designing of primers to the flanking regions of a new maize SSR is reported. The new marker, with a core motif of (TC) 12, designated as MZETC34, was mapped to the long arm of chromosome 10.